Vitamins and their metabolites are essential for a number of physiological processes including regulating the immune systems. For instance, vitamin D3 (VD3) can affect the functions of immune cells including macrophages, dendritic cells (DC), B and T cells. 25-hydroxyvitamin D3 (25(OH)VD3) is the main circulating form of VD3 whereas 1,25(OH)2VD3 is the most physiologically active form of VD3. Of interest, decreased serum levels of 25(OH)VD3 have been reported in patients with autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes mellitus (DM) and multiple sclerosis (MS). In fact, VD3 supplementation in patients with MS and mice with experimental allergic encephalitis (EAE, animal model of MS) reduced their disease. Although these findings support a role for VD3 in autoimmunity, its mechanism(s) is largely unknown. Addressing this issue is critical since it provides a scientific rationale for supplementing VD3 as well as can lead to developing new treatments in autoimmunity. Interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and forkhead box P3 (FOXP3)-positive regulatory T cells (hereafter called FOXP3+ Treg) play major roles in autoimmunity and inflammation. The Treg population likely contains two subsets, one called naturally occurring Treg developed from the thymus (FOXP3+ nTreg) and the other one induced from conventional CD4+ T cells in the periphery (FOXP3+ iTreg). Overproduction of IL-17 as well as alterations in the numbers and function of FOXP3+ Treg have been linked to animal models of various autoimmune diseases and their human counterparts. Studies suggest that the differentiation of Th17 cells and FOXP3+ iTreg from na[unreadable]ve CD4+ T cells have a reciprocal relationship. The current application address the hypothesis that 1,25(OH)2VD3 directly regulates the FOXP3 and IL-17 gene expression in human CD4+ T cells, leading to the enhanced development of FOXP3+iTreg with a reciprocal suppression of Th17 cell differentiation and IL-17 production. The goal of the application is addressing the hypothesis with the following specific aims: Aim 1) Investigate the effects of 1,25(OH)2VD3 on developing FOXP3+ iTreg and Th17 cells as well as the mechanisms underlying such effects in human CD4+ T cells;and Aim 2) Investigate the molecular mechanism underlying 1,25(OH)2VD3-mediated FOXP3 and IL-17 gene regulation through identifying and characterizing the binding site(s) of the vitamin D receptor in the human FOXP3 and IL-17 genes. The results of the proposed studies will identify a mechanistic link between autoimmunity and VD3 via FOXP3+ Treg and Th17 cells and provide a scientific rationale for supplementing VD3 in autoimmunity and developing new treatments with VD3 and its analogues. Public Health Relevance: Vitamins are known to modulate immune cells like CD4+ T cells, a type of white blood cells. The goal of the current application is to investigate the effects of vitamin D on two different CD4+ T cell subsets: T-helper 17 cells with inflammatory property and regulatory T cells with anti-inflammatory property. The results of the studies will advance our knowledge on how vitamin D can be used as a therapeutic agent to improve inflammation in humans.